Histological differentiation markers are useful in the diagnosis of carcinoma metastases where the location of the primary tumor is uncertain or unknown. Unfortunately, markers specific for a single epithelium or organ are currently available for a only few types of carcinoma, e.g., prostate-specific antigen for prostate carcinomas and thyroglobulin for thyroid carcinomas.
Less specific markers of transitional cell carcinomas have been identified and associated with malignant transformation, tumor progression and the prognosis. Many of these markers are epithelial membrane antigen (EMA) or oncogene/tumor suppressor gene products. For example, Summerhayes et al. (1985. JNCI 75:1025-1038) have described a series of monoclonal antibodies (group III), directed against the urothelium which produce luminal-membrane staining of normal superficial (umbrella) cells of the urothelium. Other markers are expressed in superficial bladder tumors but disappear in invasive and metastatic transitional cell carcinomas. All of these markers are antibodies most of which stain non-urinary epithelia and carcinomas too. Certain antigens such as involucrin, E48 antigen and SCC antigen are markers shared by both transitional and stratified squamous epithelia (of skin, esophagus, cervix, etc. ) and their carcinomas. However, no differentiation or lineage marker specific for transitional cell carcinomas and their metastases has been identified to date.
Normal urothelium contains tissue-specific differentiation products that have been well characterized morphologically and biochemically. It has been found that large numbers of urothelial plaques are present in the superficial plasma membrane of urothelial superficial or umbrella cells. These plaques are characterized by a highly unusual membrane structure, i.e., the asymmetric unit membrane (AUM), whose luminal leaflet is twice as thick as its cytoplasmic leaflet. The thickening of the luminal leaflet is due to the presence of particles exhibiting a semi-crystalline organization. The molecular constituents principally comprise four transmembrane proteins: uroplakin (UP) Ia (27 kDa); UP Ib (28 kDa); UP II (15 kDa) and UP III (47 kDa). These UPs, particularly UP Ia, Ib, and II, are characterized by their markedly asymmetric mass distribution, with the extracellular domain being considerably larger than the intracellular one. This accounts for the clearly visible ultrastructural thickening of the luminal leaflet of the unit membrane. UP III is believed to play a role in the formation of the urothelial glycocalyx and may interact, via its cytoplasmic portion, with the cytoskeleton.
The identification and characterization of the uroplakins and their role as molecular markers has been described by Yu et al. 1990. J. Cell Biol. 111:1207-1216; Wu et al. 1990. J. Biol. Chem. 265:19170-19179; Wu et al. 1993. J. Cell Sci. 106:31-43; Wu et al. 1994. J. Biol. Chem. 269:13716-13724; Yu et al. 1994. J. Cell Biol. 125:171-182; and Lin et al. 1994. J. Biol. Chem. 269:1775-1784. Further, Ryan et al. (1993. Mammalian Genome 4:656-661) describe mapping the chromosomal locations of mouse and bovine UP Ia and UP Ib and suggest likely locations for human genes. The locations of human UP II, UP IIIa and UP IIIb genes are also predicted. Ryan et al. suggest that uroplakin genes could be involved in chronic urinary tract diseases and also suggests diagnostic uses (e.g., interstitial cystitis). Mono-specific antibodies to UP Ia, UP Ib, UP II and UP III were used to show that uroplakin expression is confined to the urinary bladder and to determine the location of UP Ia, UP Ib, UP II and UP III within the asymmetric unit membrane. However, identification, sequencing, or chromosomal location of the human gene for any of the uroplakins has not yet been disclosed.
Yu et al. (1992. Epith. Cell Biol. 1:4-b 12) describe the use of specific antibodies to AE31 and AE32 for analysis of the differentiation state of bovine urothelial cells. The diagnostic use of antibodies to uroplakins to positively identify metastatic bladder carcinomas has also been described by Moll, et al. 1993. Verh. Deutsc. Ges. Path. 77 and Moll, R. et al. 1995. Am. J. Pathol. 147:1383-1397.
The human gene for uroplakin II has now been isolated and sequenced. Using the knowledge of the human uroplakin gene sequence a highly specific uroplakin-based reverse transcriptase (RT)-polymerase chain reaction (PCR) assay has now been developed which is useful in the detection of circulating bladder cancer cells in metastatic bladder cancer patients.